This project aims to find novel restriction enzymes that will be useful in many aspects of molecular biology research. One particular focus will be on restriction endonucleases that are able to recognize modified DNA, especially DNA containing 5-methylcytosine and 5- hydroxymethycytosine since such enzymes have many potential applications in epigenetics research. Enzymes recognizing these modified bases are known to occur in bacterial and archaea strains and in the past, many have been found by random biochemical screening. In the proposed project, the discovery of potential new enzymes will use bioinformatics techniques and take advantage of the large number of currently sequenced prokaryotic genomes, both completely sequenced genome and shotgun sequences. The project will identify good candidates and then use biochemical methods to characterize them. In addition to commercializing any useful enzymes that are found from this screen, the information gained about the biochemical function of the restriction enzymes discovered will be invaluable for genome annotation and will have practical implications for researchers working with pathogenic organisms. The latter will result from our efforts to characterize the restriction-modification systems in pathogens and identify those systems which need either to be knocked out or circumvented by prior modification prior to transformation. Thus, the results of this research will have immediate implications for the fields of epigenetics, pathogenesis and genome annotation.